Cytogenetics ‘2’ - KSU

CYTOGENETICS ‘2’Karyotyping, cell preparation and staining

9/8/2015NAHLA BAKHAMIS. MSc

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Chromosomes

• 1970 Banding techniques

- identification of individual chromosomes

• Karyotype and FISH

- types of abnormalities;

. Extra copy of chromosome

. Missing copy of chromosome

. Structural abnormalities


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Chromosomes • Centromere

- movement during cell division

- divides the chromosomes into short (p) and long (q) arms

• Telomere - Tip of each chromosome

- seal chromosomes and retain chromosome integrity

- consists of tandem repeats TTAAGGG

- maintained by enzyme; telomerase

- telomerase & in tandem repeats imp in aging & cell death


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Chromosomes Classified according to position of centromere ;

• Central centromere; metacentric

• Sub-terminal centromere ; acrocentric

- have satellites which contain multiple copies of genes for

ribosomal RNA

• Intermediate centromere; submetacentric


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Chromosomes

Chromosomes ideogram


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Chromosomes

• 22 autosomal, and sex chromosomes in pairs

• Classified according to:

- Length

- position of centromere

- presence or absence of satellites

• Chromosomes divided into groups labelled A-G


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Chromosomes

Chromosomes divided into groups labelled A-G


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Chromosomes


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Karyotyping

Staining method to identify chromosomes in a single cell

G banding .. Giemsa

Q banding .. Quinacrine, fluorescent stain (structural rearrangements)

R banding .. Reverse

C banding .. Centromeric (heterochromatin)

Ag-Nor stain .. Nucleolar Organizing Regions (active)


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Cell preparation

Required cell stage; metaphase

1. Culture cells until sufficient mitotic activity

2. Harvest protocol:

• Mitotic arrest

• Add hypotonic treatment

• Fixation with mix of methanol ; acetic acid

• Want long chromosomes with non-overlapping


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Harvest protocol

a. Mitotic arrest:

Colemid arrest cells at the equatorial plate by preventing

spindle fibres formation


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Mitosis

Studyblue.com


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Harvest protocol

b. Hypotonic treatment:

• Cells swell (osmotic potential):

reduce cytoplasm & allow chromosome spreading

• RBCs lysis


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Harvest protocol

c. fixation:• Prevent further swelling

• Maintain good morphology

Methamoglobin: brown supernatant formed after addition of fixative

Multiple fixative -- clear supernatant


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Harvest protocol

Harvesting procedure:

1. Add 100µ colemid to the culture 20min at 37C 2. Spin; 8 min at 1000 RPM

3. Discard supernatant, add 10ml hypotonic solution

4. Incubate 20min at 73C

5. Add 5 drops fixative 3:1 methanol:acetic acid, spin

6. Discard supernatant & resuspend pellets

7. Add 10ml fixative 30min at RT

8. Repeat until clear solution

9. Preserve at fridge (-4C) not freezer


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Dropping protocol

• 8 drops on slide

• dry in a controlled environmental evaporation chambers

• Observe mitotic index

• Aging: 60C over night Or 90C for 90min for better banding

pattern


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Dropping

• Residual cytoplasm staining quality

• Dark chromosomes with sharp borders

• No overlapping, no burst


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Natureprotocols IMSTARA.com glown.com

Burst overlapped good

Metaphase compactness


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Cell preparation


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Banding (staining)


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G banding

• Most common

• Chromosomes treated with trypsin;

- denatures protein

allow Gimsa react with exposed DNA (wang &Federoff)


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G banding Gimsa stain:

• Each chromosomes characterised by dark & light band

• 400 bands / haploid genome

• Dark bands are gene poor

• Appropriately stained chromosomes;

neither too dark nor too pale


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G banding

Under-trypsinized chromosomes:

Indistinct bands, little contrast and fuzzy


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G banding

Over-trypsinized chromosomes:

• Sharp bands and frazzled at the end

• Eventually become very pale (ghost-like) and very swollen

• Fetal calf serum immersion is recommended (α1-antitrypsin)

Broz. J et al, Scielo (2000)


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G banding

Staining procedure:

• Slides must cool down to RT• Immerse in trypsin solution 10-15 sec

• Wash in phosphate buffer or serum (stop trypsin activity)

• Transfer to Gimsa stain 90-120 sec

• Raines in d-water

• Dry

• Examine under microscope


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G banding

• Count chromosomes in 10-15 metaphases

• Count 30 if mosaicism suspected

• Detailed analysis of 3-5 metaphase


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• 13,18,21 gene poor (very dark chromosomes)

• 21 smaller than 22

• 21 (200 genes) twice as mane as 22 (400

genes)

• Bands stains darkly with Gimsa DNA rich in AT

pairs (genes poor)

• Pale bands (gene active)

G banding


28Normal Male Karyotype Normal Female Karyotype

G banding


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Q banding

• Especially used for Y chromosome abnormalities or mosaicism

• similar to G band (exp. It can detect polymorphism)

• Need fluorescent microscope


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• Used to identify X chromosome

abnormality

• Heat chromosomes before treating

with Gimsa

• Light and dark bands are reversed

R banding


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C banding

• To identify centromere/ heterochromatin

• Heterochromatic region:- contains highly repetitive DNA sequence

- highly condense chromatin fibres

- genetically inactive (structural elements)

• Treated chromosomes:

- Acid

- Alkaline

- Then G band


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Karyotyping

Organization of the chromosomes, lined up according to:

• Size

• Location of centromere

• Banding pattern


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Karyotyping

Natureprotocols.com


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Karyotyping

Natureprotocols.com


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Ideogram

Is a schematic representation of

chromosomes

Show relative size of chromosomes

& their banding patterns


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ISCN

International System for Human Cytogenetic Nomenclature

• Each area of chromosome given number

• Lowest number (proximal) to centromere

• Highest number (distal to centromere)


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ISCN


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ISCN

, 46,XX,del(5p)

separates

- chromosome numbers

- Sex chromosomes

- Chromosome abnormalities

; 46,XX,t(2;4)(q21;q24)

Separates

- Altered chromosomes

- Break points structural rearrangement involving more than 1

chromosome


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ICSN

Normal Male

46,XY


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Normal Female

46,XX


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Karyotyping activity

• Make a karyotype:

http://mrforde.blogspot.com/2009/01/karyotype-game.html

Have fun ;)

http://mrforde.blogspot.com/2009/01/karyotype-game.html


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Thank You

Documents

Cytogenetics ‘2’ - KSU